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Principles of Chromatography
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Chromatography is the most powerful tool for separating & measuring the components of a complex mixture.
Quantitative & qualitative analysis
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What is Chromatography?
1) Solvent Extraction :
transfer of a solute from phase 1 phase 2
S (in phase1) S (in phase 2)
partition coefficient
1
2
s
sK
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2) Chromatography : same as extractiona) One phase: held in place stationary phase. solid material (packing material)
Another phase : fluid phase mobile phase. sample gas (GC) liquid (LC)
What is Chromatography?
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What is Chromatography?
b) A solute equilibrates between a mobile and a stationary phase.The more it interacts with the stationary phase, the slower it is moved along a column.
Xm Xs
Ks = [X]s / [X]mSolutes with a large Ks value will be retained more strongly by the stationary phase.
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What is Chromatography?
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c) The science & art of separation
d) Originator : adsorption chromatography by M.Tswett in 1903
e) Eluent, eluate, elution.
What is Chromatography?
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elution : always (100%) dilution
What is Chromatography?
sam plein
eluentin
CaCO 3
(adsorption)
colum n
eluantout
detector
chrom atogram(m ass spect. IR
spect. etc)
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3) Types of Chromatography
Is divided into categories on the basis of the mechanism of interaction of the solute v.s. the stationary phase.
What is Chromatography?
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polar s.p.
What is Chromatography?
for GC & LC for GC
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21.1 What is Chromatography?
resin-SO3- gel filtration
resin-N(CH3)3+ by size
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What is Chromatography?
Ask Yourself 20-A p.461pH, and ionic strength
the most selective one
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How do we describe a chromatogram
1) Chromatogram :
A graph showing the detectors
response as a function of elution
time : band’s shapes, position,
resolution.
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2) For individual band :
a) Retention time (tr) :the time needed after injection for an individual solute to reach detector.
b) An ideal chromatographic peak Gaussian shape. w½ = 2.35σ, w = 4σ
How do we describe a chromatogram
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How do we describe a chromatogram
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How do we describe a chromatogram
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3) For pairs of bands
a) Efficiency : two factors contribute to how well components are separated :
the widths of the peaks :
the wider the peak, the poorer separation.
the spacing in time :
the further apart, the better separation.
How do we describe a chromatogram
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b) Theoretical plates (N): (from distillation)the more plates on a column, the more
equilibration steps, and the better the
separation.
Number of plates on column :
N = 5.55(tr/w½)2
Plate height : H = L/N
The smaller plate height
narrower peaks better separation
How do we describe a chromatogram
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c) Resolution (Rs)
How do we describe a chromatogram
Rs2 s.p. theoflength 2
1.5Rs analysis, vequantitatiFor
Lw
0.589t
ww21
tt
w
ΔtRs
1/2av
r
21
rr
av
r 12
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Qualitative: • Co-chromatography• Detector:
– Mass spectrometer– IR, UV-VIS spectrophotometer
d) Qualitative & Quantitative analysis
How do we describe a chromatogram
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• Figure illustrates the point that computers and humans may not choose the same baseline for measuring area.
P.464
Qualitative and Quantitative Qualitative and Quantitative AnalysisAnalysis
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Internal Standards
• An internal standard is known amount of a compound, different from analyte, that is added to an unknown.
• To use an internal standard, we prepare a known mixture of standard and analyte and measure the relative response of the detector to the two species. In Figure 5-6, the area under each peak is proportional to the concentration of each compound injected into the column.
P.119
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• [X] and [S] are the concentrations of analyte and standard after they have been mixed together.
P.119
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ExampleExample : : Using an Internal StandardUsing an Internal Standard• In a chromatography experiment, a solution containing
0.083 7 M X and 0.066 6 M S gave peak areas of Ax=423 and AS=347.
• To analyze the unknown, 10.0 mL of 0.146 M S were added to 10.0 mL of unknown, and the mixture was diluted to 25.0 mL in a volumetric flask.
• This mixture gave the chromatogram in Figure 5-6, with peak areas Ax=533 and AS=582. Find the concentration of X in the unknown.
P.119
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SOLUTIONSOLUTION ::
P.120
Because X was diluted from 10.0 to 25.0 mL when the mixture with S was prepared, the original concentration of X in the unknown was (25.0/10.0)(0.057 21 M)=0.143 M.
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Why do bands spread ?
1) Why broadening?a) diffusionb) slow equilibration of solute between the m.p and s.p.c) irregular flow paths.
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Why do bands spread ?
2) Longitudinal diffusion :
the faster the flow
the less a band spends in column.
the less time for diffusion.
broadeningu
1
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Why do bands spread ?
3) solute requires time to equilibrate between phases.
(s.p.m.p.) with temp. broadening u
Can’t equilibrate rapidly enough.
m.p.
s.p.
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Why do bands spread ?
Solute requires a finite time to equilibrate between the mobile and stationary phases.
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P.466
4) A Separation Has an Optimum Flow Rate4) A Separation Has an Optimum Flow Rate
• The rate of mass transfer between phases increases with temperature.
Optimum resolution (minimum plate height) occurs at an intermediate flow rate. Curves show measured plate height in gaschromatography of n-C17H36 at 175°C, using N2, He, or H2 mobile phase.
Why do bands spread ?
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Why do bands spread ?
5) Multiple paths
Band spreading from multiple flow paths. The smaller the stationary-phase particles, the less serious is this problem. This process is absent in an open tubular column.
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Why do bands spread ?
6) Plate height equation
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Plate height equation
Why do bands spread ?
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Why do bands spread ?
7) open tubular columns
Packed column (A, B, C 0 in van Deemter’s eqn.)
Open tubular column (A = 0 in van Deemter’s eqn.)
resolution (∵ H & column length) sample capacity (∵ less s.p.)
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Why do bands spread ? 8) Funny shapes
polarsolute
OH OH
SiSi S i S iOO
OSi(CH 3)3(CH3)3SiO
s.p. silanization
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Mass Spectrometry
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P.470
Mass Spectrometry• Mass spectrometry measures the masses and
abundances of ions in the gas phase.
A Mass SpectrometerA Mass Spectrometer
• Figure next page shows a transmission quadrupole mass spectrometer, which is the most common mass separator in use today.
• The mass separator consists of four parallel metal rods to which a constant voltage and a radio-frequency oscillating voltage are applied.
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Transmission quadrupole mass spectrometer.
P.470
Figure 21-13
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• Ionization: 1) Electron ionization
2) Chemical ionization
Mass Spectrometry
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1) Electron ionization
M + e- M+ + e- + e-
70 eV -55 eV 0.1eV
Molecular ion break into fragments.
Base peak: most intense peak.
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2) Chemical ionization
CH4 + e- CH4+ + 2e-
CH4+ + CH4 CH5
+ + CH3
CH5+ + M CH4 + MH+
CH4+ CH3
+ + H
CH3+ + CH4 C2H5
+ + H2
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• Total ion Chromatograms is a reconstructed total ion chromatogram showing all ions from seven opium alkaloids found in street heroin.
• Selected ion Chromatograms:– Simplify analysis – improve S/N
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P.473
Information in a Mass Spectrum
Nominal Mass : CNominal Mass : C44HH99Br is 136 Br is 136
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Information in a mass spectrum
Rxn : CH3(CH2)2CH2–OH + Br- CH3(CH2)2CH2–Br
1–Butanol 1–Bromobutane
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CH3 15
CH2 14
Br 79
C4H979Br+ 50.0%
C4H981Br+
Information in a mass spectrumFragmentation Patterns
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Information in a mass spectrumIsotope PatternsCnHxOyNz
12C/13C
Intensity = n x 1.1%
Ex: C6H6
(M+1)/M+ = 6 x 1.1 %
Nitrogen Rule: A compound: odd nominal mass / odd number of N atoms;
even nominal mass/ even number of N atoms